A series of proline analogs have been analyzed for their effects on collagen synthesis inhibition in cultures of primary DMBA-induced rat mammary tumors and for their effects on mammary tumor growth in tumor bearing animals. Azetidine carboxylate, thioproline and cis-hydroxyproline were found to be potent, selective inhibitors of collagen synthesis, blocking amino acid incorporation into collagen by 7 to 27 fold more than incorporation into total tumor cell protein. In vivo all 3 of these compounds at doses of 50-2000 mg/kg S.C., caused tumor growth arrest or regression. The conditions favoring proline analog sensitivity of mammary tumors have been assessed. A positive correlation exists between the ability of a tumor to synthesize basement membrane and its proline analog sensitivity. Sensitivity is approximately proportional to the efficacy of the analog in blocking collagen synthesis in vultured turmor epithelium. The epithelium of normal mammary glands and mammary adenocarcinomas is dependent on proline for optimal growth, especially when cells are plated on stromal collagen substrata. Blocking basement membrane deposition and thereby favoring tumor cell contact with stroma may, therefore, promote proline analog uptake and tumor cell kill. In contrast to primary tumors, metastatic rat mammary tumor growth was not affected by proline analogs. Electron microscopy revealed that the latter tumors lacked a basement membrane. Using cDNAs against type IV collagen, we have demonstrated a lack of collagen IV mRNA sequences in the metastatic tumors whereas the collagen message is present in collage IV producing, proline analog sensitive, primary tumors.